Genome-wide identification of the Q-type C2H2 zinc finger protein gene family and expression analysis under abiotic stress in lotus (Nelumbo nucifera G.)

Background Lotus (Nelumbo nucifera G.) is an important aquatic plant with high ornamental, economic, cultural and ecological values, but abiotic stresses seriously affect its growth and distribution. Q-type C2H2 zinc finger proteins (ZFPs) play an important role in plant growth development and environmental stress responses. Although the Q-type C2H2 gene family has been identified in some plants, limited reports has been carried out it in lotus. Results In this study, we identified 45 Q-type NnZFP members in lotus. Based on the phylogenetic tree, these Q-type NnZFP gene family members were divided into 4 groups, including C1-1i, C1-2i, C1-3i and C1-4i. Promoter cis-acting elements analysis indicated that most Q-type NnZFP gene family members in lotus were associated with response to abiotic stresses. Through collinearity analyses, no tandem duplication gene pairs and 14 segmental duplication gene pairs were identified, which showed that duplication events might play a key role in the expansion of the Q-type NnZFP gene family. The synteny results suggested that 54 and 28 Q-type NnZFP genes were orthologous to Arabidopsis and rice, respectively. The expression patterns of these Q-type NnZFP genes revealed that 30 Q-type NnZFP genes were expressed in at least one lotus tissue. Nn5g30550 showed relatively higher expression levels in all tested tissues. 12 genes were randomly selected with at least one gene from each phylogenetic clade, and the expression of these selected genes were confirmed by qRT-PCR (quantitative real-time polymerase chain reaction). The results indicated that Q-type NnZFP genes were extensively involved in cadmium, drought, salt and cold stresses responses. Among them, 11 genes responded to at least three different stress treatments, especially Nn2g12894, which induced by all four treatments. Conclusions These results could increase our understanding of the characterization of the Q-type NnZFP gene family and provide relevant information for further functional analysis of Q-type NnZFP genes in plant development, and abiotic stress tolerance in lotus. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-024-10546-1.


Background
Plant growth and development has been threatened by various abiotic stresses [1], such as drought, salinity, extreme temperatures, and heavy metals.Transcription factors (TFs) act as critical regulators involved in various biological and environmental stress processes through transcriptional regulation of downstream genes in plants [2].Zinc finger proteins (ZFPs) are one of the largest families of TFs in plants, and they can self-fold into a "finger" structure by binding to Zn 2+ [3].ZFPs can be classified into different categories based on the position and quantity of His and Cys residues, including C4HC3, C3H, C3HC4, C4, C2H2, C2HC, C6, and parallel types [4].Among them, C2H2 ZFPs consist of two Cys and two His residues that coordinate with a zinc atom, forming a zinc finger structure with a β-sheet and an α-helix [5].
The Q-type ZFP subfamily with "X 2 -C-X 2 -C-X 3 -F-X 3 -QALGGH-X 3 -H", belongs to the C2H2 ZFPs family [6,7], and they have been isolated from various plant species, such as petunia [8], Arabidopsis [9], poplar [10], rice [11], wheat [12], and potato [13].These Q-type ZFPs play vital roles in growth and various abiotic stress responses in plants [14][15][16][17].For example, the expression of StZFP1 in potato increases under salt and drought stress, and ectopic expression of StZFP1 in tobacco enhances salt tolerance of transgenic plants [18].In addition, overexpression of AtZAT10 improves cold resistance in Arabidopsis [19], and overexpressing OsZFP245 increases drought tolerance in rice [20].PuZFP103 from poplar enhances drought tolerance by regulating the levels of substances such as MDA, EL, proline, and soluble sugars [21].AtZAT6 enhances cadmium tolerance in Arabidopsis through specifically binding to the promoter of GSH1 and regulating glutathione synthesis [22].However, these studies have mainly focused on model or terrestrial plants, with limited reports in aquatic plants.
Lotus (Nelumbo nucifera G.) is a perennial aquatic plant belonging to the Nelumbonaceae family.It is one of the traditional flowers in China with high ornamental, economic, cultural and ecological values, and very popular cultivated worldwide nowadays [23].In addition, lotus is a basal eudicot plant with numerous monocot characteristics, so it is also an important subject for evolutionary and taxonomic studies [24].However, adverse environment stresses such as extreme temperatures, salinity, heavy metals and drought stresses greatly affect lotus survival and growth, including leaf yellowing and wilting, petal decayed, and disruption of the redox balance system, photosynthesis system, and cellular structure [25][26][27].These studies are mainly limited to phenotype, physiological and biochemical characteristics of lotus responses to environment stresses, but the exact molecular mechanism of stress resistance in lotus remains largely unknown.Therefore, the exploration of stress-responsive genes relate to lotus is of great importance for its broader applications.To date, most of the reports relate to stress-responsive transcription factors of lotus have focused on bHLH [24], WRKY [28], WUSCHEL-related homeobox (WOX) [29], and so on.However, no lotus Q-type ZFP family genes participate in various abiotic stress have yet been identified.
In the current study, we firstly identified Q-type NnZFPs according to the genome of lotus, and then analyzed their phylogenetic relationships, sequence features, chromosome distribution, gene duplications and cis-regulatory elements.In addition, the expression profiles of these Q-type NnZFP genes in various tissues and their responses to abiotic stresses were investigated.These results will provide valuable information in predicting the roles of the Q-type NnZFP gene family in lotus.Moreover, the study could lay the foundation for creating resistant resources of lotus through molecular breeding.

Plant materials and treatments
The seeds of lotus Weishanhuhonglian were provided by Weishanhu Hedu Aquatic Flower Breeding Base (Shandong, China).The seeds were germinated in distilled water with the top part of the blunt end pierced.And they were cultured for next 3 weeks at 30 ℃ with a long-day photoperiod of 16 h light/8 h dark, and a light intensity of 12,000 lx.After three weeks, healthy and uniformly growing seedlings were selected for different abiotic stress treatments, including drought stress treatment (20% polyethylene glycol, PEG6000), salt stress treatment (300 mmol/L NaCl), cadmium stress treatment (30 μmol/L CdCl2), and low temperature treatment (4℃).In contrast, seedlings were cultured in distilled water as a control.Five treatment time points (0, 6, 12, 24 and 48 h) were performed.The experiment was performed with three biological replicates.Leaf samples from the treated and control seedlings were collected, frozen in liquid nitrogen, and stored at -80 °C for subsequent RNA extraction.

Database sources and identification of Q-type NnZFPs in lotus
The protein sequences of the Q-type C2H2 ZFP gene family in Arabidopsis thaliana L. were downloaded from database (https:// www.arabi dopsis.org/) according to a published article [1].All these protein sequences were queried to search the lotus Q-type C2H2 family through local protein blast (BLASTP) against the whole genome sequence database (http:// nelum bo.biocl oud.net), with an E value ≤ 1 e−5 .After removing redundant sequences, the candidates were further submitted to SMART (http:// smart.embl-heide lberg.de/) and the NCBI Conserved Domain Database CDD-search (https:// www.ncbi.nlm.nih.gov/) was used to manually screen Q-type C2H2 ZFP members in lotus for further analysis.

Analysis of phylogenetic, gene structure and conserved composition of Q-type NnZFPs in lotus
The number of amino acid, molecular weight and isoelectric point of Q-type NnZFPs were analyzed using the online tool Expasy (http:// web.expasy.org/ compu tepi/).Subcellular localization was predicted using the WoLF PSORT website (https:// wolfp sort.hgc.jp/).The phylogenetic tree was constructed based on the alignment of amino acid sequences for Arabidopsis Q-type ZFPs and lotus Q-type ZFPs members by MEGA11 with the maximum likelihood method and 500 replicate bootstrap tests.The MEME 5.5.0 software (http:// meme-suite.org/ tools/ meme) was used to predict protein conserved motifs, with the following parameters: maximum number of motifs: 10; and the optimum motif widths: 5-150 amino acid residues.The analyses of exon-intron structures of Q-type NnZFP genes were carried out by comparing the coding sequences with their corresponding protein sequences.Then the map of phylogenetic tree, conserved motifs, and intron-exon structures of Q-type NnZFPs were visualized and merged using TBtools [30].

Analysis of chromosomal distribution, gene duplication and cis-regulatory element
The chromosome distribution information of each Q-type NnZFP gene was acquired from the genome annotation data of lotus.Based on TBtools, the chromosomal positions map of Q-type NnZFP genes and the relative distances were obtained.MCScanX software was used to analyze the duplication events and synteny of the Q-type ZFPs between lotus and Arabidopsis, and lotus and rice.The resultant microsynteny relationships were further evaluated by CollinearScan set at an Evalue of < 1 e−5 , and the figures were drawn by TBtools [30].In addition, the ratio of non-synonymous to synonymous nucleotide substitutions (Ka/Ks) was evaluated among duplicated gene pairs to detect the selection mode by Ka/Ks Calculator and ParaAT2.0.The sequences of 2000 bp from the promoter region of these Q-type NnZFP genes were extracted using TBtools [30].Then the PlantCARE database (http:// bioin forma tics.psb.ugent.be/ webto ols/ plant care/ html/) was used to identify the cis-regulatory elements of these Q-type NnZFPs.Among them, we chose 8 cis-elements that were most related to abiotic stress, hormones and plant growth, including TGT CGG / TGT CTC (AuXREs), MYB-binding sites (MBSs), abscisic acid response elements (ABREs), G-boxes (LREs), CGTCA/ TGACG-motif (JAREs), dehydration-responsive element/ C-repeat (DRE/CRT), TCA-elements, WRE and WUNmotif.The results were finally visualized using TBtools [30].

Tissue specific expression of Q-type NnZFP genes
Based on genome-wide transcriptome data downloaded from the lotus Genome Database (http:// nelum bo.cngb.org/ nelum bo/ tools/ expre ssion Visua lizat ion) [31], the expression profiles of Q-type NnZFP genes in various tissues (apical bud, seed coat, cotyledon, root, rhizome internode, immature receptacle, mature receptacle, leaf, immature stamen, mature stamen, petal, petiole, pollinated carpel, unpollinated carpel, rhizome elongation and rhizome apical meristem) from all developmental stages were explored [31].These data and the IDs of these Q-type NnZFPs were uploaded to the "HeatMap" procedure in TBtools [30].In control dialog, lower and higher levels of transcript accumulation were indicated by red and blue, respectively, and the median level was signaled by white, the expression heatmap was generated subsequently.

RNA isolation and quantitative real-time PCR
A total of twelve Q-type NnZFP genes were randomly selected as candidates and the synthesized primers in Table S1 were designed by primer premier 5. Total RNA were extracted with the RNA extraction kit (Vazyme, NanJing, China).Elimination of genomic DNA contamination and first-strand cDNA synthesis were carried out using the PrimeScript ™ RT reagent Kit with gDNA Eraser (Vazyme, NanJing, China).The qRT-PCR analyses of the expression of each gene under different stress conditions were performed using the 2 × Taq Master Mix ChamQ Universal SYBR qPCR Master Mix kit (Vazyme, NanJing, China) with the QuantStudioTM Real-Time PCR system (QuantStudio5, Applied Biosystems, Hammonton, NJ, USA).The programs were performed as the following steps: 95 °C/30 s for predenaturation (step 1), 95 °C/10 s for denaturation (step 2), 60 °C/30 s and 95 °C/15 s for primer annealing and extension (step 3), then go to step 2 for 40 circles, and the collection program of melting curve was default in the system.The obtained data were analyzed using the 2 −△△Ct method [32], with NnActin (LOC104593066) serving as the internal control [33].

Statistical analysis
All statistical analyses were calculated with SPSS version 18.0 (SPSS Inc., Chicago, IL, USA).Statistical differences between measurements on different treatments were determined by Students t-test for significance analysis (*: p<0.05; **: p<0.01).All measurements were performed with three biological replicates and three technical replicates of each biological replicate, and data were expressed as mean ± standard deviations (SD).

Identification and physicochemical properties of Q-type NnZFPs
The BLASTP algorithm search was performed against the lotus genome, and then we manually selected the Q-type ZFP members with the specific sequence "X 2 -C-X 2 -C-X 3 -F-X 3 -QALGGH-X 3 -H".Finally, a total of 45 Q-type ZFPs in lotus were obtained after screening domains and removing redundant genes.Physicochemical analyses of the 45 Q-type NnZFPs revealed that they ranged from 145 to 590 amino acids in length.The molecular weights of these proteins were varied from 16,179.64 to 65,646.06, and their isoelectric point (pI) ranged from 5.02 to 9.27.Moreover, subcellular localization prediction indicated that the majority of Q-type NnZFP members (34/45, 75.6%) were predicted to be localized in the cell nucleus, while only 11 members showed potential localization in other cellular compartments, including peroxisomes, extracellular matrix, chloroplasts, endoplasmic reticulum, plasma membrane, and cytoplasm (Table S2).

Phylogenetic analyses and classification of Q-type NnZFPs
To clearly investigate evolutionary relationships, a phylogenetic tree was constructed based on the alignment of amino acid sequences for 56 Arabidopsis Q-type ZFPs and 45 lotus Q-type ZFPs members by MEGA11 with the maximum likelihood method and 500 replicate bootstrap tests.According to the phylogenetic tree (Fig. 1), the Q-type ZFP subfamily were divided into four clades, namely C1-1i, C1-2i, C1-3i, and C1-4i.Twenty-nine NnZFPs and twentyeight AtZFPs were assigned to the C1-1i group, seven NnZFPs and eighteen AtZFPs belonged to the C1-2i group, seven NnZFPs and eight AtZFPs were grouped into the C1-3i group, and C1-4i group possessed two NnZFPs and two AtZFPs.

Gene structure and motif composition analyses of the Q-type NnZFPs
The protein motifs are highly conserved amino acid residues, which are considered to possibly have functional and structural roles in active proteins.To reveal the diversification of the identified Q-type NnZFPs, their full-length protein sequences were inspected for 10 conserved motifs according to the phylogenetic tree (Fig. 2A).As shown in Fig. 2B, motif 1 and motif 4 were widely discovered in almost all Q-type NnZFPs.Moreover, other motifs were specific to specific groups.For example, members of the C1-1i commonly contained motif7 and motif9.In the C1-2i group, certain members possessed motif2 and motif5.Groups C1-3i and C1-4i displayed a similar motif composition, including motif3, motif6, motif8 and motif10.
The evolutionary process could be forecasted by analyzing the gene structures.In order to have deep insight into gene structures of these Q-type NnZFPs, we analyzed the number of exons and introns.The results showed that the number of introns varied from 0 to 5.There were 39 members (86.7%) without introns, and they all belonged to the C1-2i, C1-3i and C1-4i groups.while the C1-1i group contained six members with 1-5 introns, with Nn3g17856 and Nn4g23857 processing 4 and 5 introns, respectively (Fig. 2C).These results indicated that members within the same group of Q-type NnZFPs exhibited similar gene structures, but differences existed in members among different groups.

Chromosomal distributions and gene duplications in Q-type NnZFPs
Members of the Q-type NnZFPs in lotus were unevenly distributed across 8 chromosomes.Among them, chromosome 2 had the highest number of genes, with a total of 11 genes, while only 1 gene was located on chromosome 8 (Fig. 3).
Gene duplication lead to generate a large number of novel genes.In the present study, no tandem duplication gene pairs and 14 segmental duplication gene pairs (26/45,57.78%)were identified using MCScanX methods (Fig. 4).Among them, Nn1g02680 and Nn2g11224 were both collinear with two genes.In addition, we calculated that the ratio of Ka/Ks of Q-type NnZFP duplication gene pairs varied from 0.0849 to 0.3465 (Table S3).
To further understand the evolutionary relationship of Q-type NnZFPs, we analyzed the synteny relationship of Q-type ZFP genes among lotus, Arabidopsis, and rice.The results showed that a total of 54 pairs of orthologs were identified between lotus and Arabidopsis (Fig. 5A), and the Ka/Ks ranged from 0.0720 to 0.3791 (Fig. 5C, Table S4).Furthermore, 28 pairs of orthologs between lotus and rice were identified (Fig. 5B), with the Ka/Ks varied from 0.1051 to 0.3216 (Fig. 5C, Table S5).In addition, the results indicated that 16 and 8 Q-type NnZFP genes were homoeologous to multiple genes in Arabidopsis and rice, respectively.Thus, the results suggested that the Q-type NnZFP genes in lotus had higher similarity with Q-type AtZFP genes in Arabidopsis.

Analyses of Q-type NnZFP gene promoter cis-regulatory elements
Cis-regulatory elements play key roles in the transcriptional regulation for precise initiation and efficiency of genes.To better understand the regulatory network of Q-type NnZFP genes, we analyzed the promoter regions of Q-type NnZFP genes.And 8 cis-elements (AuXREs, MBSs, ABREs, LREs, JAREs, DRE/CRT, TCA-elements, WRE and WUN-motif.)that are most related to abiotic stress, hormones and plant growth were explored.According to the Figure S2 and Table S6, MBSs were widely distributed in the promoters of most of Q-type NnZFP genes.Considering MYB as key TFs involved in regulatory networks controlling plant development and responses to abiotic stresses, the large distribution of MBSs might enhance the role of the Q-type NnZFP genes in regulating the growth and development of the lotus and coping with the external environment stresses.In addition, 7 Q-type NnZFP genes (e.g.Nn1g04591, Nn2g10467, etc.) have a large number of ABREs in their promoters, so we speculated that these genes have similar functions with ABA-responsive factors.Taken together, our results suggested that these Q-type NnZFP genes may respond to different pathways through different types of cis-acting elements within their promoter regions.

Analyses of tissue specific expression of Q-type NnZFP genes
To understand the tissue-specific expression patterns of the Q-type NnZFP genes, the transcript abundances in different tissues from all developmental stages (apical bud, seed coat, cotyledon, root, rhizome internode, immature receptacle, mature receptacle, leaf, immature stamen, mature stamen, petal, petiole, pollinated carpel, unpollinated carpel, rhizome elongation and rhizome apical meristem) of lotus [31] were analyzed (Fig. 6).The results showed that 30 (66.7%) Q-type NnZFP genes were expressed in at least one lotus tissue.Among them, Nn5g30550 showed relatively high expression levels in all tested tissues with log 2 FPKM > 1, indicating that Nn5g30550 played an essential role in lotus growth.Additionally, some Q-type NnZFP genes performed a tissue-specific expression pattern.For example, Nn2g12696 was only highly expressed in seed coats, and Nn1g04656 exhibited high expression levels

Expression analyses of Q-type NnZFP genes in lotus under abiotic stresses
To probe the transcript abundance patterns of Q-type NnZFP genes under cold, drought, salt and heavy metal stresses, 12 genes were randomly selected with at least one gene from each phylogenetic clade.And the expression of the selected genes were investigated by qRT-PCR.
Under Cd stress, 12 Q-type NnZFP genes showed different responses (Fig. 7). 4 genes were highly expressed under Cd stress, including Nn6g35222, Nn1g04591, Nn2g12894, and Nn2g10467.Among them, Nn6g35222 and Nn1g04591 expression levels immediately increased and remained relatively high levels compared with the control after 6 h of Cd treatment.By contrast, the expression levels of Nn2g12894 and Nn2g10467 gradually increased and reached the maximum at 24 h before a gradual decrease.Notably, the largest increase in expression was detected for Nn2g12894, which was 12 times more than the control.5 Q-type NnZFP (Nn7g35949, Nn2g15752, Nn2g12286, Nn4g24490, and Nn3g18442) transcripts were down-regulated by Cd stress since 6 h.Different from the above-metioned genes, Nn5g30550, Nn2g13856, and Nn6g33594 showed no significant changes in expression between CK and Cd treatment.
The changes of Q-type NnZFP gene expression under salt stress can be divided into three situations (Fig. 8).Firstly, compared with CK, six genes were induced by salt stress, including Nn6g35222, Nn5g30550, Nn2g13856, Nn1g04591, Nn2g12894, and Nn2g10467.Among them, Nn2g12894 exhibited the largest increase (23-fold) in expression at 24 h by salt stress.Secondly, the expression levels of 5 genes (Nn7g35949, Nn2g15752, Nn2g12286, Nn4g24490, and Nn3g18442) were lowered under NaCl treatment than those in CK.Lastly, Nn6g33594 maintained relatively stable expression levels after salt treatment compared with control, similar to its expression patterns under Cd stress.
For the drought treatment, two genes (Nn2g13856 and Nn2g12894) were significantly up-regulated compared to CK (Fig. 9).They both reached their maximum expression levels at 12 h with 2.8 and 4.3 times that of the control, respectively.Compared to CK, expression levels of 7 genes (Nn6g35222, Nn5g30550, Nn7g35949, Nn2g15752, Nn2g12286, Nn4g24490, and Nn3g18442) were down-regulated under drought treatment.In addition, after PEG treatment, there were three genes with stable expression levels similar to CK, including Nn1g04591, Nn2g10467, and Nn6g33594.

Discussion
TFIIIA-type ZFPs, known as C2H2-ZFPs, were firstly reported in the last century during the study of amphibian oocyte cells [3].Q-type ZFPs are a subfamily of C2H2-ZFPs and play essential roles in plant growth and development, and responses to abiotic stresses.To date, Q-type ZFP gene family has been identified in many species.For example, there are 56 members in Arabidopsis [3], 47 members in wheat [34], 25 members in cabbage [35], and 58 members in alfalfa [36].Similarly, a total of 45 Q-type NnZFPs were identified through lotus genome in this study.The genome size of the five plants was different, with 125 Mb in Arabidopsis [37], 17 Gb in wheat [38], 530 Mb in cabbage [39], 3.15 Gb in alfalfa [40] and 929 Mb in lotus [31].Thus, the number of Q-type ZFP superfamily members may be relatively stable in plants and it has no absolute correlation with genome size.
The number of introns and exons can affect the functional evolution of genes [41].Structural analyses of the Q-type ZFP subfamily showed that 86.7% of Q-type NnZFP genes had no introns (Fig. 2C).Low intron numbers of genes accelerate its process of transcriptional expression, and it is convenient to decrease the cost for transcription and make cell a fast reaction to abiotic stresses [42].As a result, these Q-type NnZFP genes may play an important role in the corresponding stress of lotus.The motif composition analysis of Q-type NnZFPs revealed that all 45 members contained motif1, which was identified as the core sequence specific to Q-type NnZFPs (Fig. S1A) and played an important role in DNA binding [43].Additionally, motif4, identified as the ethyleneresponsive element binding-factor-associated amphiphilic repression (EAR) motif, was present in 42 out of the 45 members (93.3%) (Fig. S1B).The EAR-motif is one of the most dominant transcriptional repression motifs identified in plants.To date, many Q-type ZFPs containing the EAR-motif reported in plants are recognized as transcriptional repressors [44].For example, the LATE FLOW-ERING (LATE) gene (At5g48890) and the KNUCKLES (KNU) (At5g14010) are demonstrated as transcriptional repressors of cellular proliferation in Arabidopsis thaliana L. [45,46].In addition, the EAR-motif transcription factor ZAT12 was demonstrated to be function as a negative regulator for iron (Fe) deficiency responses through its direct interaction with the bHLH protein FIT in Arabidopsis [47].These results revealed that the Q-type NnZFPs are rich in potential transcriptional inhibitors.But extensive researches have also shown that some Q-type NnZFPs have positive regulatory effects.For example, GsGIS3 enhances tolerance to Al toxicity through positive regulation of Al-tolerance-related genes [48], and OsZFP15 is a positive regulator of ABA catabolism and thus accelerates seed germination [49].
Gene duplication contributes to the amplification of new gene family members [50], and some evolving new members may lose their original functions or acquire new functions to enhance plant adaptability or become pseudogenes [51].In our research, no tandem duplication was found and 14 segmental duplication pairs (26/45, 57.78%) were identified (Fig. 4, Table S3).This result suggested that the expansion of the Q-type NnZFP gene family might be mainly caused by the segmental duplication.A Ka/Ks value of 1 denotes neutral selection, Ka/Ks less than 1 suggests purification selection and Ka/ Ks more than 1 indicates positive selection [52].The Ka/ Ks ratios of all duplication genes pairs in this work varied from 0.0849 to 0.3465, suggesting that the Q-type NnZFP genes might have experienced purifying selection in the process of evolution.These duplicated Q-type NnZFP gene pairs were the results of natural and artificial selections during species evolution, and it is important to investigate their duplication relationship for further researches on the evolutionary relationship of this gene family in lotus.
Q-type C2H2 ZFPs are widely involved in the plant growth and development, including seed germination and maturation [53], leaf development [15], and floral organ development [17].In the present study, Nn5g30550 was observed to be preferentially expressed in all tested organs (Fig. 6), suggesting a key role of this gene in regulating lotus growth and development.Two genes named AtAZF1 and AtAZF2, homoeologous to Nn5g30550 (Table S4), were demonstrated to affect the expression of many ABA-repressive genes in plants and severely affected seedling growth [54].Additionally, some Q-type NnZFP genes performed a tissue-specific expression pattern.For example, Nn6g35222 was highly expressed during petal development (Fig. 6), indicating that it may be associated with floral organ development.It has been reported that At3g10470, which was homoeologous to Nn6g35222 (Table S4), affected male gametophyte development in Arabidopsis [55].Furthermore, Nn2g12696 was only highly expressed in seed coats (Fig. 6).The phylogenetic analyses showed Nn2g12696 was clustered with AtZFP3 (At5g25160), which could influence seed germination by interfering with ABA and light signaling [56].These results may lead to more directed understanding the function of these Q-type NnZFP genes in lotus development biology.
Plant growth is frequently threatened by environmental stresses, such as drought, low temperature, salt, and heavy metals [57][58][59][60].Many stress-related Q-type ZFPs can help plants adapt to these abiotic stresses.For instance, GhZAT6 could resist salt stress in G. hirsutum by regulating ROS-related gene expression [61]; JcZAT10, which was regulated by ZAT12 and CBFs in Jatropha curcas, controlled the expression of low-temperature-related COR genes, thereby enhancing its cold resistance [62].In our study, a total of 12 Q-type NnZFP expression patterns under abiotic stresses were explored using qRT-PCR, and we found most of these Q-type NnZFP transcripts changed under different stress treatments.According to the expression profiles in Figs. 7, 4 genes were up-regulated under Cd treatment, including Nn6g35222, Nn1g04591, Nn2g12894, and Nn2g10467, suggesting the involvement of them in responses to Cd stress.To date, limited researches have focused on the role of Q-type NnZFPs in plant Cd tolerance.As far as we known, AtZAT6 (At5g04340) is demonstrated to improve Cd tolerance in Arabidopsis through specifically binding to the promoter of GSH1 and regulating glutathione synthesis in Arabidopsis [22], and AtZAT10 (At1g27730) was demonstrated to play dual roles in cadmium uptake and detoxification in Arabidopsis [63].Generally, members within one cluster may have common evolutionary origins and conserved functions [64].Nn6g35222, Nn1g04591, Nn2g12894, and Nn2g10467.wereclustered with AtZAT6 and AtZAT10 in C1-2i in our findings, indicating that they may have a positive role in Cd tolerance in lotus.Moreover, 6, 2, 4 genes were induced by salt, drought and cold stresses, respectively (Fig. 8-10).Similarly, AtZAT18 was demonstrated to be involved in drought tolerance in Arabidopsis [65], and OfZAT35 is an important regulator of cold tolerance in transgenic tobacco [66].We also found that there is a large overlap of Q-type NnZFP genes has multiple roles in three or more stresses (Fig. 7-10).For example, Nn2g13856 and Nn2g10467 could be induced by Cd, NaCl and PEG treatment at the same time.Specially, we found that Nn2g12894 was induced by all the four treatments.It may be related to the fact that this gene containing multiple stress and hormone related ciselements in its promoter, such as JARE, AuxRE, LREs, CBF and MBSs.These results indicated that these genes carried out multiple physiological and biochemical functions to faced environmental challenges.Therefore, the expression patterns of Q-type NnZFP genes under various abiotic stresses provided many new candidate genes for further exploration of the mechanisms of resistance in lotus.

Conclusion
In the current research, we performed a comprehensive analysis of Q-type NnZFPs family in lotus.A total of 45 Q-type NnZFPs were identified in the lotus genome and unevenly mapped on 8 chromosomes.These Q-type NnZFPs were divided into four groups, including C1-1i, C1-2i, C1-3i and C1-4i.Segmental duplication events played a key role in the expansion of the Q-type NnZFP gene family and these gene pairs evolved under strong purifying selection.Synteny analyses indicated that 54 and 28 NnZFP genes were orthologous to Arabidopsis and rice, respectively, suggesting that the Q-type NnZFP genes have higher similarity with Q-type AtZFP genes.In addition, their expression profiles in various tissues and their development stages and responses to Cd, NaCl, drought and cold stress conditions demonstrated that this gene family was widely involved in lotus organ development and abiotic stress responses.All of these results will help to reveal the biological functions of Q-type NnZFP genes, and provide a basis direct for further functional analysis of these genes in growth and abiotic stress tolerance in lotus.

Fig. 1
Fig. 1 Phylogenetic analysis and classification of Q-type NnZFPs and Q-type AtZFPs.The phylogenetic tree represents the relationship between 45 Q-type ZFP genes of lotus and 56 Q-type ZFP genes of Arabidopsis.All genes were clustered into four clades

Fig. 2
Fig. 2 Phylogenetic relationship, conserved motifs, and exon/intron structures of Q-type NnZFP genes.A Multiple alignment of 45 full-length amino acid sequences of Q-type NnZFP genes executed by ClustalW.The phylogenetic tree was constructed using MEGA 11.0 with the maximum likelihood method.B Schematic representation of the conserved motifs identified by MEME 5.5.0.Each colored box represents a motif and black lines represent non-conserved sequences.C Exon/intron structures of Q-type NnZFPs.Exon/intron structures were analyzed by the Gene Structure Display Server.Exons/introns of each subgroup are represented by blue boxes and black lines

Fig. 3
Fig. 3 Genomic distributions of 45 Q-type NnZFP genes across the 8 lotus chromosomes.In the "Q-type NnZFP genes distribution map" of 8 lotus chromosomes, the green bars represent each chromosome, and the black lines indicate the position of each Q-type NnZFP gene

Fig. 4
Fig. 4 Synteny analysis of Q-type NnZFP genes in lotus.Grey lines represent all synteny blocks in the lotus genome.Red lines indicate the duplicated Q-type NnZFP gene pairs in lotus

Fig. 5
Fig. 5 Comparative physical mapping showing the orthologous relationships of Q-type NnZFP genes with (A) Arabidopsis and (B) rice.The red line represents gene pairs that are homoeologous.C Ka, Ks, and Ka/Ks values of homoeologous gene pairs among species

Fig. 6
Fig. 6 Heatmap representation of the expression patterns of Q-type NnZFPs among different tissues from all developmental stages.Lower and higher levels of transcript accumulation are indicated by red and blue, respectively, and the median level is indicated by white.Note: AB: Apical Bud; SC: Seed Coat; C: Cotyledon, R: Root; RI: Rhizome Internode; IR: Immature Receptacle; MR: Mature Receptacle; L: Leaf; IS: Immature Stamen; MS: Mature Stamen; P: Petal; Pt: Petiole; PC: Pollinated Carpel; UC: Unpollinated Carpel; RE: Rhizome Elongation; RAM: Rhizome Apical Meristem

Fig. 7
Fig. 7 Expression analysis of Q-type NnZFP genes in lotus under Cd treatment, revealed by qRT-PCR.Shown are means ± standard deviations for three biological replicates and three technical replicates of each biological replicate (*: p<0.05; **: p<0.01)

Fig. 8
Fig. 8 Expression analysis of Q-type NnZFP genes in lotus under NaCl treatment, revealed by qRT-PCR.Shown are means ± standard deviations for three biological replicates and three technical replicates of each biological replicate (*: p<0.05; **: p<0.01)

Fig. 9
Fig. 9 Expression analysis of Q-type NnZFP genes in lotus under PEG treatment, revealed by qRT-PCR.Shown are means ± standard deviations for three biological replicates and three technical replicates of each biological replicate (*: p<0.05; **: p<0.01)